Abstract

Liposome-based nanoSized Particles with Incorporated Nitroxides, or nanoSPINs, were designed for EPR applications as pH probes in biological systems. Phospholipid membrane of the liposomes with incorporated gramicidin A showed selective permeability to a small analyte, H+, while protecting entrapped sensing nitroxide from biological reductants. An application of the pH-sensitive nanoSPIN in an ischemia model in rat heart homogenate allows for monitoring ischemia-induced acidosis while protecting encapsulated nitroxide against bioreduction.

Introduction

Nitroxyl radicals (NRs) are very useful functional EPR probes allowing measurement of redox state,1 oxygen,2 pH,3–5 NO,6–8 thiols,9–11 and structural studies of biological macromolecules.12 However, comparatively fast reduction of the NRs to EPR-silent hydroxylamines limits their application. NRs encapsulation in the inner aqueous phase of inert nanospheres, such as lipid vesicles, may result in the development of stable paramagnetic probes. The semipermeable membrane of these nanospheres prevents chemical interactions of the probe with the reducing microenvironment, while retaining the probe's ability to monitor small analytes (e.g. NO or H+). We term these paramagnetic sensors nanoSPINs (nanoSized Particles with Incorporated Nitroxides). Swartz et al. were the first to demonstrate that NRs incorporated in liposomes13 or proteinaceous microspheres14 have higher resistance to bioreduction and might be useful probes for in vivo EPR oximetry. Later our labs8,15 and others16 used encapsulation of NO-sensitive NRs in phospholipid liposomes to protect extremely unstable nitronylnitroxides against bioreduction. In this paper we describe the pH-sensitive nanoSPINs based on liposomal encapsulation of the imidazoline NRs.

Imidazoline NRs were found to be the most effective spin pH probes due to the presence of protonatable N-3 atom in the vicinity to the N−O• radical fragment.17 While a wide variety of pH-sensitive imidazoline NRs have been synthesized, the development of more stable probes remains a critical step for their successful applications, particularly in vivo.18,19

Materials and methods

Chemicals

pH-sensitive membrane-impermeable NRs were synthesized as described below. Egg α-phosphatidylcholine and L-glutathione were purchased from Sigma-Aldrich. Gramicidin A was purchased from CALBIOCHEM. Triarylmethyl free radical, TAM Oxo63, was a gift from Nycomed Innovations Co (Malmö, Sweden).

Synthesis of trimethylammonium methyl sulfate, nitroxide NR1

The nitroxide NR1 was prepared through alkylation of 4-(2-aminoethylamino)-1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazole, a2, according to the Scheme 1.

Synthesis of the nitroxide NR2

The nitroxide b7 was prepared from 3-hydroxyamino-3-ethylpentan-2-one hydrochloride b118 using a modified general procedure.21,22 The nitroxide b7 was then treated with methane sulfonyl chloride to give methanesulfonate b8, a highly reactive nitroxide. The latter undergoes nucleophilic substitution of methanesulfonate group with chloride anion upon acidification with hydrochloric acid to give b9. The nitroxide b9 is stable in the form of hydrochloride and can be used as alkylating pH-sensitive spin label.

Synthesis of the nitroxide NR2 from b9 precursor

NR2 was synthesized through the alkylation of glutathione with 2-(4-(chloromethyl)phenyl)-4-dimethylamino-1-oxyl-2,5,5-triethyl-2,5-dihydro-1H-imidazol b9. Nitroxide b9 (6.8 mg, 20 μmol) was added to a solution of glutathione (10 mmol, 1 mL) and pH was brought to 11 by adding NaOH. The reaction mixture was allowed to stay for 5 h. The unreacted nitroxide b9 was extracted with chloroform (3 × 0.7 mL). Yield of the nitroxide NR2 (3.1 mM, 1 mL), determined by EPR, was 31%. The NR2 has distinguishable EPR spectrum from NR b9 reflecting longer rotation correlation time in agreement with its larger molecular weight (Mw = 607 and the ratio of amplitudes of central- and high-field spectral components, Ic/Ih = 1.6 for the NR2; Mw = 336 and Ic/Ih 1.2 for the NR b9). Mass spectrum of the NR2-H+ (M+): calcd. for C28H44N6O7S 608.29, found 608.3.

Liposomes preparation

Liposomes preparation by extrusion

Large (200 nm diameter) unilamellar liposomes from egg phosphatidylcholine (PC) were prepared by slightly modified extrusion method described previously24 using LiposoFast extruder (Avestine, Inc., Ottava, Canada). A mixture of the ethanol solutions of PC (50 mg, 0.5 mL) and gramicidin A (25 μM, 40 μL) was dried on the walls of rotating cylinder under the nitrogen flow and then kept under the vacuum for 0.5 h. For EPR experiments, the lipid film was hydrated by moderate shaking for 1 h in 1 mL of 70 mM phosphate buffer, pH 7.2, containing l mM NR. The resulting suspension was passed through the extruder. In order to remove spin label from outer liposomal volume, the liposome suspension was passed through the gel-filtration column (Sepharose CL2B, 10 × 0.8 cm) equilibrated with the same buffer and liposome fraction was collected.

Preparation of liposomes by reverse-phase evaporation

Large unilamellar liposomes were prepared by slightly modified reverse-phase evaporation approach described previously.25 PC (45 mg) was dissolved in chloroform (3 mL). A solution of gramicidin A (25 μM, 40 μL) in chloroform was added. The mixture was placed to a 50 mL round-bottom flask with a long extension neck, and the solvent is removed under reduced pressure by a rotary evaporator. Then lipid was redissolved in the diethyl ether, and 1 mL of 1–2 mM solution of the NR in 50 mM phosphate buffer, pH 7.4, was added. The resulting two-phase system is sonicated for 5 min in a bath-type sonicator (Branson 3510) until the mixture becomes a homogeneous opalescent dispersion that does not separate for at least 30 min after sonication. The sonication temperature is 0–5 °C. The mixture is then placed on the rotary evaporator and the organic solvent is removed under reduced pressure. The liposomes were purified from the nonencapsulated NR by passing through a Sephadex G25 column.

The prepared liposome-based nanoSPINs were characterized and used on the day of preparation. The leakage of the probe from inner space of the liposomes was monitored by addition of membrane-impermeable paramagnetic broadening agent, potassium ferricyanide, and was less than 10% after 24 h incubation at room temperature.

A heart was excised from a Sprague-Dawley rat (about 300 g) and kept in liquid nitrogen before homogenization. The heart was homogenized in the presence of an equal volume of NaCl isotonic solution. The homogenate was centrifugated at 4 °C, 8000 g and supernatant was collected for further experiments.

The oxygen-sensitive EPR probe, triarylmethyl radical Oxo63, was used to measure oxygen consumption in the rat heart homogenate after addition of succinate. The dependence of the EPR linewidth of Oxo63 probe on oxygen concentration was calibrated in 0.1 M phosphate buffer, pH 7.0 and 37 °C, saturated by various nitrogen–oxygen gas mixtures using a Temperature and Gas Controller (Noxygen, Germany).

The EPR spectra of the nitroxide NR2 and encapsulated NR2 were measured in separate experiments in the presence of homogenate after addition of 10 mM succinate at 37 °C. The decay of the integral intensity of the EPR signal and the nitrogen hyperfine splitting, aN, measured as the distance between low-and high-field spectral components, were used to calculate NR concentration and pH of the sample, respectively. The temperature of the sample during the experiment was controlled by a Temperature and Gas Controller (Noxygen, Germany).

EPR measurements

EPR measurements were performed in 50 μL capillary tubes using a Bruker X-band EMX spectrometer. Parameters of the acquisition were as follows: microwave power, 20 mW; scan time, 20.97 s; modulation amplitude, 1.0 G for the NR1 and 1.5 G for the NR2. For the Oxo 63 TAM probe the spectra acquisition parameters were as follows: microwave power, 0.6 mW; modulation amplitude, 0.1 G; scan time, 20.48 s.

Characterization of the synthesized compounds

The IR spectra were recorded on a Bruker Vector 22 FT-IR spectrometer in KBr pellets (the concentration was 0.25%; the pellet thickness was 1 mm).

The UV spectra were measured on a HP Agilent 8453 spectrometer in EtOH.

The NMR spectra were recorded on a Bruker AV 300 (300.132 MHz for 1H and 75.476 MHz for 13C) spectrometer for 5–10% solutions at 300 K using the signal of the solvent as the standard. The assignment of the signals in the 13C NMR spectra was made based on analysis of intensities, on the spectra measured in J-modulation mode.

Schematic design of pH-sensitive nanoSPINs. The phospholipid membrane of the liposomes with incorporated gramicidin A showed selective permeability to a small analyte, H+, while protecting the entrapped sensing nitroxide from biological reductants.

Both NR1 and NR2 were found to be localized in the inner aqueous space of the liposome as confirmed by addition of membrane-impermeable paramagnetic broadening agent, potassium ferricyanide, to the bulk solution of the liposomes (see Fig. 2 for the NR1). No significant leakage of the NRs was observed during several hours of exposure.

Fig. 3 displays the EPR spectra of the NR1 at various pH. The EPR spectrum measured at pH close to pKa of the NR represents superposition of the spectra of protonated, NRH+, and nonprotonated, NR, forms of the radicals as clearly seen from the high-field spectral components in Fig. 3B. The ratio [NRH+]/[NR] reversibly varies with pH according to the Henderson–Hasselbalch equation providing an experimental tool for pH determination by EPR. In general, spectral simulation is required for accurate [NRH+]/[NR] determination. In practice, nitrogen hyperfine splitting measured as a distance between unresolved spectral components can be used as a highly sensitive pH marker as shown in Fig. 4.

pH dependences of nitrogen hyperfine splitting, aN, for the NR1 and NR2 in aqueous solution (○) and in gramicidin-containing liposomes (●). Solid lines correspond to the best fit of experimental data to standard titration equation yielding...

Liposomes prepared without the gramicidin pore sustained a transmembrane pH gradient of several units of pH.27,28 Incorporation of gramicidin A channels resulted in dissipation of the transmembrane proton gradient allowing for the monitoring of extraliposomal pH by the encapsulated spin pH probe. Indeed, similar pH dependences of aN were obtained for the NR free in solution and encapsulated in liposomes (see Fig. 4).

Fig. 5 illustrates the protective effect of the liposomal encapsulation of the NR2 against a physiologically relevant reducing agent, ascorbate. Insignificant, about 5%, loss of EPR signal, apparently attributed to slow transmembrane diffusion of ascorbic acid,29 was observed in the presence of 10 mM ascorbate (100 fold excess of the ascorbate over the NR) after 25 min of incubation. Note that EPR signal of the NR2 free in solution was practically undetectable after 25 min incubation in the presence of significantly lower, 1.5 mM, concentration of ascorbate (see Fig. 5).

We applied NR2 encapsulated in gramicidine-containing liposomes to monitor ischemia-induced acidosis in the rat heart homogenates. The oxygen consumption by homogenate oxidative metabolism was measured by EPR using oxygen-sensitive probe Oxo63. As shown in Fig. 6C oxygen level dropped down below detectable level 10 min after succinate addition. During this time the encapsulated NR2 kept 85% of its signal intensity. On the other side, EPR signal of the NR2 added to homogenate free in solution was practically undetectable after 10 min of incubation (see Fig. 6A). Therefore, use of NR2 containing nanoSPINs allowed us for accurate measurement of the aN and calculation of corresponding pH values shown in Fig. 6B.

Previously we applied pH-sensitive imidazoline NR with similar range of pH sensitivity (pK 6.1) to monitor ischemia-induced acidosis in isolated rat heart using L-band EPR-spectroscopy.5 However, the loss of about 90% of the EPR signal intensity of the NR incubated in the heart during 30 min of global ischemia limits its application. The pilot experiment using the encapsulated NR2 in the ischemic rat heart shows four times higher stability of the EPR signal (data not shown), therefore supposing pH-sensitive nanoSPINs to be a useful tool for the studies of myocardial acidosis.

Conclusion

In this work we formulated the concept of a stable paramagnetic nano-sized analytical probe, nanoSPIN, and designed the first liposome-based nanoSPINs for pH monitoring in biological systems using EPR spectroscopy. NanoSPINs significantly enhance the stability of the nitroxide probes, and may become useful analytical tools, particularly taking into account the importance of pH status in various physiological and patho-physiological processes, e.g. extracellular pH in tumors30,31 and ischemia-induced miocardial acidosis. Further improvements in stability of the liposomes may be achieved using archaebacterial lipids32,33 or UV-induced cross-linking polymerization of lipid monomers.34–36 An alternative strategy to nanoSPIN design may be based on preparation of capsules with a polyamide membrane37 used for encapsulation of enzymes or preparation of artificial cells.38 Recent developments of PEBBLEs (nanosized Photonic Explorers for Bioanalysis with Biologically Localized Embedding)39 and polymer nanocapsules40 demonstrate alternative strategies for encapsulation of molecular probes within an inert matrix which can be explored for the design of the nanoSPINs.

Acknowledgements

This work was partly supported by NIH grants 1R01 GM072897, KO1 EB03519, and R21 CA132068 and RFBR grants 08-03-00432-a and 08-04-00555.